Recently, as the fact that various diseases are caused by the abnormal activity of cell proteins is known, the development of drugs capable of adjusting the activity of such proteins to treat fatal human diseases becomes the object of interest in the whole world.
In a description of the present invention, superoxide dismutase related to reactive oxygen species is mentioned as one example of the cell proteins. However, it is to be understood that the scope of the target proteins in the present invention is not limited only to this protein.
Reactive oxygen species are inevitably produced as byproducts of intracellular metabolism in all living beings where energy is obtained using oxygen. Such reactive oxygen species cause damage to biopolymers, such as intracellular protein, nucleic acid and fat, and have a deep connection with the progression of various diseases of the human body. Particularly, they are involved in carcinogenesis processes, apoplexy, arthritis, radiation damage and inflammatory reaction, and act as an important factor of promoting aging even in a normal aging process [Floyd, R. A., FASEB J. 4, 2587-2597 (1990); Anderson, W. F., Human gene therapy, Nature 392, 25-30 (1998); and Halliwell B. and Gutteridge J. M. C., Free radicals in biology and medicine, Oxford University Press, Oxford (1999)].
Diseases related to Cu/Zn-superoxide dismutase are summarized in Table 1 below.
TABLE 1CategoriesConcrete examplesInflammatory/Glomerulonephritis, vasculitis, autoimmuneimmune damagediseases and the likeIschemiaStroke, myocardial infarction, arrhythmia,angina pectoris and the likeDrug and toxic—substance-inducingreactionIron overloadIdiopathic hemochromotosis and the like(tissue and serum)Radiation-related damageNuclear bombing, radiation therapy and the likeAgingProgeria, and disease-related agingRed blood cellsSickle-cell anemia, malaria and the likeBronchiA result of smoking, emphysema and the likeHeart andCardiomyopathycardiovascular systemsKidneysAutoimmune nephrotic syndromeStomach and intestinesBetelnut-related oral cancerAbnormal conditionsHypoxia, Alzheimer's disease, Parkinson'sof brains/nervousdisease and the likesystem/nervous musclesEyeCateract and the likeSkinDiseases caused by UV irradiation, and the like*Quoted from “Free radicals in biology and medicine”, Oxford University Press, Oxford, pp 618-619.
Known reactive oxygen species include 1O2, OH, O2, H2O2 and the like. They are produced by various enzymatic reactions, and play an important role in the biosynthesis and immune function of various physiologically active substances, and drug metabolism. However, if they are over-produced by external radiation, UV, environmental pollution and various stresses, they can rather cause damage to the living body. For this reason, the living body has enzymes, such as SOD, catalase and peroxidase, for their defensive function, and if its aging is started, the balance of the skin will be upset and the ability of this enzyme to protect the skin from various reactive oxygen species will be reduced.
Thus, a need to protect the skin from such reactive oxygen species is being increased, and SOD, lactoferrin, and antioxidants, etc., are used or developed as raw materials of cosmetics for the removal of the reactive oxygen species. However, SOD was not advantageously employed in a cosmetic composition in spite of its ability to remove the reactive oxygen species, because it has the nature of enzymatic protein and thus problems in that it is difficultly soluble in lipid, has insufficient stability, and is impermeable to the skin's keratin layer due to its molecular weight of more than 30,000 Daltons.
Cu/Zn-superoxide dismutase is an important intracellular defensive enzyme of preventing the cellular damage caused by free radical toxicity and oxygen-radical damage [Fridovich, I., Annu. Rev. Biochem., 64, 97-112 (1995)]. Since all polymers in the living body are always exposed to this harmful action of the oxygen radical, an interest to use the Cu/Zn-superoxide dismutase for the treatment of various diseases is being increased.
Recently, there are many attempted methods for clinically applying the Cu/Zn-superoxide dismutase. Methods for delivering the Cu/Zn-superoxide dismutase into the living body, which have been developed till now, can be broadly classified into the following three categories. First, there is a method of conjugating polyethylene glycol, ficoll, lecithin, albumin and the like to the Cu/Zn-superoxide dismutase [Del Zoppo, G. J. et al., Drugs 54, 9-38, (1997); and Muzykantov, V. R. et al., Proc. Natl. Acad. Sci. USA 93, 5213-5218, (1996)]. Second, there is a method of encapsulating the Cu/Zn superoxide dismutase with liposome [Perdereau, B. et al., Bull. Cancer 81, 659-669 (1994)]. Third, there is a genetic therapy where the Cu/Zn superoxide dismutase gene is transduced into cells to induce the overexpression of the enzyme in the cells [Okumura, K. et al., Pharm. Res. 14, 1223-1227; (1997); Lehmann, T. G. et al., Transplantation 69, 1051-1057 (2000); and Liu, R. et al., Hum. Gene Ther. 8, 585-595 (1997)].
Among such methods, the genetic therapy is most attracted, and many studies to use the Cu/Zn-superoxide dismutase gene for the treatment of diseases have been conducted. However, the genetic therapy has various problems in that a method of delivering the gene into a cell is not easy, the percent expression of the gene in a target cell is low, and it is very difficult to artificially adjust the amount of expression of the protein in the target cell [Verma, I. M. et al., Nature 389, 239-242 (1997)].
As another method of delivering a therapeutic drug or protein into a cell, a method of directly delivering a target protein through a cell membrane to a cell can be contemplated. However, the therapeutic drug or protein is very difficult to pass through the cell membrane due to its size or various biochemical properties. It is generally known that substances with a molecular weight above 600 are almost impossible to pass through the cell membrane.
It was recently found that a Tat (transactivator of transcription) protein as a kind of human immunodeficiency virus type-1 proteins is efficiently passed through the cell membrane such that it is easily delivered into a cytoplasm. This function appears due to the property of a protein transduction domain as the middle domain of the Tat protein, and its precise mechanism is yet unknown [Frankel, A. D. and Pabo, C. O., Cell 55, 1189-1193 (1988); Green, M. and Loewenstein, P. M., Cell 55, 1179-1188 (1988); Ma, M. and Nath, A., J. Virol. 71, 2495-2499 (1997); and Vives, E., Brodin, P. and Lebleu, B., J. Biol. Chem. 272, 16010-16017 (1997)]. However, it seems that a certain receptor or carrier is not involved in the passage of the Tat protein through the cell membrane, and this passage of the Tat protein is caused by the direct interaction between the protein transduction domain of the Tat protein and the lipid double layer of the cell membrane [Vives, E. et al., J. Biol. Chem. 272, 16010-16017 (1997); and Derossi, D. et al., J. Biol. Chem. 271, 18188-18193 (1996)].
Recent studies showed that when heteroproteins, such as ovalbumin, β-galactosidase, and horseradish peroxidase, was administered in a form fused with an HIV-1 Tat protein, they were delivered directly into each tissue of the living body and a cultured cell [Fawell, S. et al., Proc. Natl. Acad. Sci. USA 91, 664-668 (1994); Schwartze, S. R. et al., Science. 285, 1569-1572 (1999); and Watson, K. and Edward, R. J., Biochem. Pharmacol., 58, 1521-1528 (1999)]. This test result suggests that the Tat protein has the ability to deliver itself and also other macromolecules into the cell.
However, it is not that substantially all proteins are delivered by the Tat protein. Furthermore, whether all the proteins delivered by the Tat protein into the cell show biological activity is not yet certainly established.
Moreover, the present inventors conducted an intracellular transduction test for the following fusion proteins: a fusion protein where a HIV Tat protein transduction domain (residues 49-57) is covalently bonded to the amino-terminal end, a fusion protein where an oligolysine transduction domain having 6-12 lysines are covalently bonded to the amino-terminal end; and a fusion protein where a basic transduction domain from which 2 or 3 residues of HIV Tat residues 48-57 had been deleted, are covalently bound to the amino-terminal end. The test results showed that the fusion proteins had the ability to be smoothly transduced into the cell (Korean patent laid-open publication Nos. 2002-10446 and 2002-67108).